Thalassemia is the name given to a group of inherited blood disorders characterized by abnormal hemoglobin production. Hemoglobin is the oxygen-carrying molecule of red blood cells. The dominant hemoglobin in adult humans (hemoglobin A) is comprised of four protein chains (or globins) including two α-chains (or α-globin) and two β-chains (β-globin). Other types of globin in two minor forms of hemoglobin include γ-globin and δ-globin. If a person's body does not produce enough of these protein chains, red blood cells do not form properly resulting in a condition known as anemia, or a deficiency of functional red blood cells.
Thalassemia is usually divided into four types: α, β, δβ and δ. Among them, α and β are the main types of thalassemia. The severity of α-thalassemia and β-thalassemia depends on how many of the four genes coding for α-globin and the two genes coding for β-globin are missing or mutated, respectively. The human α-globin gene cluster is located on the short arm of chromosome 16 (cytogenetic location: 16p13.33) and comprises the HBA1 (α1-globin) and HBA2 (α2-globin) genes. The human β-globin gene cluster is located on the short arm of chromosome 11 (cytogenetic location: lip 15.4) and comprises the HBB gene (β-globin gene).
Thalassemia is more prevalent in tropical and sub-tropical regions. See U.S. Pat. No. 6,322,981, the content of which has been incorporated herein by reference in its entirety, for a further discussion of the prevalence of thalassemia around the world. Thus, those afflicted with thalassemia are often of Asian, African, Mediterranean, or Middle Eastern descent. In China, thalassemia is prevalent in provinces south of the Yangtze river including Guangxi, Guangdong, Guizhou, Hainan, Yunnan, and parts of Sichuan.
Among Chinese patients, α-thalassemia is often caused by α-globin gene deletions such as the large fragment 3.7 kb deletion (−α3.7 or −a3.7), the large fragment 4.2 kb deletion (−α4.2 or −α4.2), and the multi-gene Southeast Asia-deletion (−−SEA or −−SEA) and Thai-deletion (−−THAI or −−THAI). See U.S. Pat. No. 5,750,345, the content of which has been incorporated herein by reference in its entirety, for a further discussion on −α3.7, −α4.2, and −−SEA deletions.
In addition, some of the more common α-globin gene point mutations among Chinese α-thalassemia patients include the αConstantSpringα (αCSα or αCSα), αQuongSzeα (αQSα or αQSα), and αWestmeadα (αWSα or αWSα) mutations. The αCSα mutation is caused by a terminator codon mutation of the α2-globin gene which results in reduced α-globin chain synthesis. The αQSα mutation is caused by a mutation of the α2-globin gene whereby the amino acid leucine of codon 125 is substituted by proline. The αWSα mutation is caused by a mutation of the α2-globin gene whereby the amino acid histidine of codon 122 is substituted by glutamine. In all such cases, these mutations result in reduced or defective α-globin chain synthesis.
In addition, β-thalassemia among Chinese patients is often caused by β-globin gene mutations including the following common mutations: (1) a frame-shift codon 41/42 (−TCTT) deletion mutation (also referred to as a 41-42M or a CD41-42M/N mutation), (2) a −28M/N (A-G) (also referred to as a −28M mutation), (3) a CD71/72 (+A) (also referred to as a 71−72M insertion mutation), (4) a CD17 (A-T) (also referred to as a 17M mutation), (5) a BEM/N (also referred to as a βEM mutation), (6) an IVS-2-654 (C-T) (also referred to as a 654M/N or a 654 M mutation). Other less common β-globin mutations include: (1) a −31 (A-G) deletion mutation (also referred to as a 31M mutation), (2) a 14-15M insertion mutation, (3) a CD 43 (G-T) (also referred to as a 43M/N or a 43M mutation), (4) a 27/28M insertion mutation, (5) an IVS-I-1M mutation, (6) an IVS-1-5 (G-C) (also referred to as an IVS-1-5M) mutation, (7) a CAPM deletion mutation, (8) an IntM mutation, (9) a −30M mutation, (10) a −29M/N (also referred to as a −29M mutation), (11) a −32M/N (also referred to as a −32M mutation), (12) a CD37M point mutation, (13) a 90M point mutation, and (14) an IVS-II-5M point mutation. See Luo, Hong-Cheng, et al. “Impact of genotype on endocrinal complications of Children with Alpha-thalassemia in China.” Scientific Reports 7 (2017).
Since α-thalassemia is often caused by large genomic fragment deletions while β-thalassemia is often caused by point mutations, different diagnostic methods have been developed for each type of disorder. For example, most laboratories and hospitals currently use breakpoint polymerase chain reaction (PCR) or Gap-PCR followed by agarose gel electrophoresis to diagnose α-thalassemia and use reverse dot-blot hybridization to diagnose β-thalassemia. However, both methods are labor intensive, involve more than ten operational steps between the two methods, and require almost two to three days to complete. In addition, the risks of contamination are high as amplified PCR tubes must be opened as part of both methods.
Other common diagnostic methods for diagnosing α-thalassemia and β-thalassemia include high resolution melting (HRM) analysis, multiplex PCR, and multiplex ligation-dependent probe amplification (MLPA). However, all such methods are also inefficient or time-consuming and lack specificity and accuracy. In addition, current methods also often require that DNA be extracted from the patient's blood rather than other bodily fluids or samples.
Therefore, a solution is needed which reduces the number of operational steps needed to complete a diagnosis for α-thalassemia and β-thalassemia yet maintains or improves the level of accuracy of such multi-step procedures. Moreover, such a solution should also reduce the amount of time needed to make an accurate diagnosis from between two to three days to less than two hours. In addition, such a solution should also lessen the risk of contamination by not necessitating that amplified PCR tubes be opened as part of the diagnostic procedure. Furthermore, such a solution should work equally well with DNA extracted from a patient's blood as DNA extracted from other bodily fluids or samples including amniotic fluid, samples derived from chorionic villus sampling (CVS), and samples derived from swabs.